Molluscicidal activity of Nicotiana tabacum extracts on the invasive snail Pomacea canaliculata

Botanical molluscicides for controlling the invasive snail Pomacea canaliculata have attracted worldwide attention because of their cost and environmental friendliness. Aqueous extracts from discarded tobacco leaf (Nicotiana tobacum) were evaluated for molluscicidal activity against different-sized P. canaliculata under laboratory conditions. The results showed that over 90% of the snails died in 1 g/L tobacco extract within 4 days, and the survival of P. canaliculata was inversely proportional to the snail size, tobacco extract concentration and length of exposure time. Adult males were more susceptible to tobacco extract than females. The snails had few chances to feed or mate in 0.5 g/L tobacco extract, and reproduction was greatly limited in 0.2 g/L. The growth of juvenile snails was inhibited in 0.2 g/L tobacco extract, but adults were unaffected. The antioxidant capacity of P. canaliculata in response to tobacco extract can be size- and sex-dependent, and the activities of superoxide dismutase, catalase, and acetylcholinesterase and the contents of glutathione and malondialdehyde were increased in adult males. These results suggest that discarded tobacco leaves can be useful as a molluscicide for controlling the invasive snail P. canaliculata based on its effects on survival, behaviour, food intake, growth performance and antioxidant capacity.


Materials and methods
Experimental materials. All experimental research was conducted in accordance with relevant institutional, national, and international guidelines and legislation. Pomacea canaliculata snails were collected from irrigation ditches in farmland near Shaoguan University, Shaoguan, China (24° 78′ N, 113° 67′ E), and identified as P. canaliculata according to the mitochondrial COI gene sequence 14 . The snails were reared in two 135-L rectangular aquaria and fed fresh lettuce Lactuca sativa L. under sunlight for approximately 1 week before we used them in this experiment. The tested snails were divided into three classes according to shell height: 5-15 mm (small), 15-25 mm (medium) and 25-40 mm (large). The snails were considered to be sexually mature if their shell heights were greater than 25 mm 15 , and shell height was defined as the horizontal distance between the top and base of the shell 16 . Male snails can be easily distinguished from females once they reach the age of maturity by observing the operculum 17 , so large snails were divided into two groups: adult males and females. Discarded flue-cured tobacco leaf (N. tabacum cv. K326) was provided by a farmer who exploits tobacco-rice rotation in Shixing County, Shaoguan City, China (25° 02′ N, 114° 15′ E) that permitted by Tobacco Research Laboratory, South China Agricultural University. And the experiments were performed in accordance with regulations of the Guangdong Tobacco Shaoguan Company. The collected tobacco leaves were dried at 60 °C for 12 h considering their moisture content in storage and then smashed into powder by a grinder before the experiment. Specimen (voucher no. 2021-03-SGSX) was stored at − 20 °C refrigerator in South China Agricultural University (contact person: Huaiyuan Li, email: leehy@scau.edu.cn). We confirm that this complies with national guidelines and no formal ethics approval was required in this case. The nicotine content of tobacco was determined using the spectrometric method (ISO 2881-1992).
Experimental procedure. Fifty grams of tobacco powder was mixed with 1 L of dechlorinated tap water in a conical flask, and then the flask was kept at 60 °C for 24 h in a water bath. A stock solution of 50 g/L aqueous tobacco extract was prepared after filtration through a two-layer gauze. Different concentrations of tobacco extract were obtained by diluting the stock solution with dechlorinated tap water to 1 and 2 g/L concentrations. Moreover, 0 g/L tobacco extract (dechlorinated tap water) served as the control group (CK). Thirty-six plastic cases were used for the survival study, which were placed in a room. Ten actively crawling P. canaliculata of each type (small, medium, adult male, adult female) were placed in each plastic case (25 × 14 × 13 cm) containing 2 L of tobacco extract, and each concentration (0, 1, 2 g/L) was tested in triplicate, so 90 individuals of P. canaliculata for each type were used. During the experimental period, the snails were not fed, and water was kept at an average temperature of 25 ± 2 °C. Each case was covered with a perforated lid to prevent the escape of snails. The survival rates of P. canaliculata of each type were assessed at 24, 48, 72 and 96 h after they were introduced into the tobacco extracts. The snails were confirmed dead if a rotten smell was detected or the operculum was unable to contract after a light touch 2 . The dead snails were removed from the cases immediately once found to avoid water pollution.
The trial found that the snails were short-lived in tobacco extracts over 1 g/L, the lethal effect of lower concentrations (0.2 and 0.5 g/L) of tobacco extract on P. canaliculata within 15 days was investigated under laboratory conditions in subsequent experiment. Moreover, the effects of tobacco extracts on the behaviour, feeding, growth, antioxidant enzyme and acetylcholinesterase (AChE) activities of P. canaliculata were measured. Nine same-sized plastic cases with perforated lids containing 2 L of tobacco extract were used as in the previous experiment for small snails [three replications per concentration (0, 0.2, 0.5 g/L)]. The survival of the snails was checked daily, and their behaviour was observed at 1, 2, 3, 4, 6, 9, 12, and 15 days after ten preweighed small snails were released in each case. The water temperature fluctuated between 28 and 30 °C during the experiment. The individual behaviour of the snails was classified into five states of activity: feeding, crawling, motionless (operculum open), closed operculum and crawling out of water. The behaviour was defined as feeding if the snails eat and crawl simultaneously. Preweighed fresh lettuce was added to each case daily and the leftovers were weighed again after 24 h to estimate the amount ingested by snails, the daily intake per snail was obtained by the lettuce consumption divided by the number of living snails. Dead snails were removed from their respective cases once found, and water was replaced every 2 days. The live snails were weighed again after being released for 15 days to calculate their growth in response to tobacco extracts.
The initial weight (W 0 ) of individual snails was in agreement with the precision of the scale (0.1 mg) and was obtained by the total weight of the snails in each case divided by the number of living individuals. The snails were allowed to crawl over a plastic surface for a few minutes to expel the water retained in the pallial cavity before weighing 18 . The terminal weight (W t ) was measured in the same way after 15 days. The weight growth rate (WGR) was calculated by Eq. (1), the feeding rate (FR) was calculated by Eq. (2), the food conversion ratio (FCR) was calculated by Eq. (3), and the specific growth rate (SGR) was calculated by Eq. (4): www.nature.com/scientificreports/ where t is 15 days and F is the sum of the daily food intake per snail within 15 days. A similar experiment was conducted for medium snails, ten preweighed individuals were released in each case and nine cases has been used, but the volume of water used was 3 L to prevent the living space from being too small. For adult snails, nine rectangular aquaria (56 × 45 × 35 cm) containing 30 L of tobacco extract (0, 0.2, 0.5 g/L) were used, and ten males and ten females were weighed and introduced into each aquarium. Each aquarium was covered with a 1.5-mm mesh plastic net to prevent the escape of snails. The water temperature was approximately 27.9-30.2 °C. The feeding and growth of adult snails were investigated in the same way, but mating behaviour was incorporated into the behavioural observations for adult snails based on the experimental design for juvenile snails. All snails were transferred to a − 20 °C refrigerator after weighing and prepared for antioxidant enzyme and AChE activity measurements.
The snails were defrosted, and the hepatopancreas was dissected. Two hepatopancreases from small snails were aggregated into one sample because the tissue from a single hepatopancreas was not sufficient to meet the amount of tissue required for testing. For the other types of P. canaliculata (medium, adult male and adult female), one hepatopancreas constituted one sample. The antioxidant enzyme and AChE activities in the hepatopancreas of P. canaliculata were measured with four replicates for small, medium and adult female snails based on surplus individuals surviving in 0.2 g/L tobacco extract and with three replicates for adult males, one sample represented a replicate. The hepatopancreas was homogenized with precooled physiological saline (0.7% NaCl, 1:9 w/v) and centrifuged at 3000 rpm and 4 °C for 10 min. Supernatants were used for determination of enzymes. The activities of superoxide dismutase (SOD), catalase (CAT), and AChE and the contents of protein, glutathione (GSH), and malondialdehyde (MDA) in the hepatopancreas of P. canaliculata were determined using commercially available assay kits (Jiancheng, Ltd, Nanjing, China) in strict accordance with kit instructions. Data analysis. The concentrations of tobacco extracts that killed 50% (LC 50 ) of P. canaliculata snails were estimated by Karber's method 19 . All the data were analysed using SPSS software version 20.0 for Windows (IBM Inc., U.S.A.). For the P. canaliculata survival rate under different tobacco extract concentrations within the same snail type and time, data were tested for normality using the Shapiro-Wilk test followed by Duncan's multiple comparison test; arcsine transformation was tried or the nonparametric Kruskal-Wallis and Nemenyi tests were used if the data failed to meet a normal distribution or equal variance was not assumed. The effects of snail size and sex and tobacco extract concentration on the survival rate of P. canaliculata were examined according to repeated-measures ANOVA with time as a within-subject factor, followed by Duncan's multiple comparison test. The effects of snail size and tobacco extract concentration on snail behaviour were assessed using repeatedmeasures ANOVA. The multiple comparison for the effect of tobacco extract concentration on food intake of P. canaliculata was explored using the same approach as that for survival rate, but the data transformation mode was changed from arcsine to logarithmic. The effects of tobacco extract concentration on weight and other feeding indicators of P. canaliculata were investigated using independent samples t test after logarithmic transformation. Differences in the antioxidant enzyme and AChE activities of P. canaliculata between the control and 0.2 g/L tobacco extract treatments were evaluated by independent samples t test, and the Mann-Whitney U test was used if the data were nonnormally distributed. Significance was considered at P < 0.05, and high significance was considered at P < 0.01.

Results
Effects of tobacco extract on the survival of P. canaliculata. All P. canaliculata of different sizes survived at 0 g/L, except for 10% of medium-sized snails that died during the experimental period ( Fig. 1; Supplementary Table 1). The survival rates of small snails subjected to various concentrations of tobacco extract were not significantly decreased compared with the control group within 48 h (P > 0.05), but the survival rate decreased to 60.0% at 2 g/L tobacco extract after 72 h that were significantly lower than the corresponding control (P < 0.05), the survival rates of small snails were 30.0% and 13.3% after exposed to 1 g/L and 2 g/L tobacco extracts for 96 h, respectively (Fig. 1a). Compared with small snails, the medium snails were more sensitive to tobacco extracts and showed lower survival rates at 1-2 g/L concentrations after 24 h; 6.7% of medium snails survived at 1 g/L tobacco extract, and all died at 2 g/L tobacco extract within 4 days (Fig. 1b). Relative to juveniles (small and medium snails), 1-2 g/L tobacco extracts caused lower survival rates in adult snails on each investigation day, and all adult snails died under 1 g/L and 2 g/L tobacco extract within 96 h and 72 h, respectively, including males and females (Fig. 1c,d). Specifically, more than 90% of different-sized P. canaliculata closed their operculum after exposure to 1-2 g/L tobacco extract. www.nature.com/scientificreports/ The repeated-measures ANOVA revealed marked effects of snail size, tobacco extract concentration, time and their two-and three-dimensional interaction on the survival rates of P. canaliculata (regardless of sex; snail size: F 2,27 = 50.024, P < 0.001; tobacco extract concentration: F 2,27 = 154.297, P < 0.001; time: F 3,81 = 189.455, P < 0.001). The size of P. canaliculata was inversely proportional to its survival rate in response to the tobacco extracts, and there was a significant difference among the three classes of P. canaliculata (P < 0.05). Similarly, the higher the concentration of tobacco extract was, the lower the survival rate of P. canaliculata, and significant differences were found between different tobacco extract concentrations (P < 0.05).
The survival rate of adult male P. canaliculata was decreased at 0.2 g/L tobacco extract and fell to 23.3% after 15 days, which was markedly lower than that of the control group from the 5th to 15th day (P < 0.05, Fig. 2c). Beyond that, more than 80% of P. canaliculata of all sizes survived after exposure to 0.2 g/L tobacco extracts for 15 days, and their survival was not affected compared with the control group (P > 0.05, Fig. 2). All snails died within 10 days at a higher concentration of tobacco extract (0.5 g/L), including small, medium, and adult males and females of P. canaliculata ( Fig. 2; Supplementary Table 2). Repeated measures ANOVA revealed that the survival rate of P. canaliculata was inversely proportional to the snail size, tobacco extract concentration and length of exposure time, and these three factors had significant effects on the survival of the snails (P < 0.05, Fig. 2). In addition, sex had a significant effect on the survival rate of snails under tobacco stress (excluding juveniles; F 1,12 = 37.538, P < 0.001) and female snails had a greater capacity to survive than males after exposure to the tobacco extracts (Fig. 2c,d).
By combining two molluscicidal assay results, the LC 50 values of tobacco extract for small, medium, adult male and adult female P. canaliculata after 48 h of treatment were 0.97, 1.04, 0.51 and 0.55 g/L, respectively. The LC 50 values of tobacco extract for small, medium, adult male and adult female P. canaliculata after 72 h of treatment were 0.80, 0.93, 0.49 and 0.41 g/L, respectively.
Effects of tobacco extract on the behaviour of P. canaliculata. Figure 3 and Supplementary Table 3 shows the behaviours displayed by the different size classes of P. canaliculata in the different concentrations of tobacco extract. Significant effects of tobacco extract concentration (F 1,12 = 5.394, P < 0.05) and snail size (F 2,12 = 20.261, P < 0.001) on feeding behaviour of P. canaliculata were observed. Only one small snail exhibited feeding ability in the 0.5 g/L tobacco extract on the 2nd day, and the remaining snails were unable to feed under this condition. In contrast, the snails consumed lettuce at 0-0.2 g/L tobacco extracts most of the time, but the adult snails showed a lower proportion of feeding behaviour than small and medium snails (P < 0.05). The tobacco extracts exhibited a significant effect on the crawling and motionless behaviour of the snails (P < 0.01), but the effect of snail size was not significant (P > 0.05). With an increase in tobacco extract concentration, more www.nature.com/scientificreports/ snails reduced crawling and stayed motionless. Few snails displayed the closed operculum behaviour during the experiment and seemed not to be affected by 0.2-0.5 g/L tobacco extracts (P > 0.05), but this phenomenon occurred more commonly in adults. Individual snails crawled out of the water occasionally during the experiment, which was not related to tobacco extract concentration and snail size (P > 0.05). Excluding juvenile P. canaliculata, the tobacco extracts significantly inhibited the copulation behaviour of the snails (P < 0.05). A few snails were found mating in the 0.2 g/L tobacco extract on the 1st day, and no copulatory behaviour was noted in the following observation, including in the snails in the 0.5 g/L tobacco extract.
Effects of tobacco extract on the feeding and growth of P. canaliculata. The snails had difficulty eating food after exposure to 0.5 g/L tobacco extract, and the food intake of different-sized snails was less than 1 g per individual during the experiment, which was much lower than that in the 0 and 0.2 g/L tobacco extracts (P < 0.05, Table 1, Supplementary Table 4). However, the amounts of food consumed by small snails were not significantly different between the 0.2 and 0.5 g/L tobacco extracts (P > 0.05). Snails of all sizes could obtain food at a lower tobacco extract concentration (0.2 g/L), but their intake was suppressed relative to the control group, except for small snails (P < 0.05, Table 1). The WGR, FR, FCR and SGR of small snails were restricted in response to tobacco stress (P < 0.05). However, for medium snails, the FR and FCR were not significantly inhibited in the 0.2 g/L tobacco extract group compared with the control group (P > 0.05); however, their growth (WGR and SGR) was significantly slowed (P < 0.05, Table 1). For adult individuals, their feeding rate was markedly inhibited in 0.2 g/L tobacco extract (P < 0.05), but their growth (WGR and SGR) was slightly higher than that of the control group, which may have benefited from their food conversion rate being dramatically increased under the stimulation of tobacco extract (P < 0.05, Table 1).
Effects of tobacco extract on the antioxidant enzyme and AChE activities of P. canaliculata. The antioxidant enzymes (SOD, CAT, GSH and MDA) and AChE activities of small snails and adult females had no significant change after exposure to 0.2 g/L tobacco extract for 15 days compared with the control group (P > 0.05, Table 2, Supplementary Table 5). In contrast, all antioxidant indices and AChE activity of adult males were significantly increased in 0.2 g/L tobacco extract (P < 0.05). For medium snails, the SOD activity and GSH content were significantly increased under tobacco stress (P < 0.05); however, no significant changes in CAT, MDA or AChE activities were observed (P > 0.05, Table 2).  Table 1. Effect of tobacco extract on the feeding and growth of P. canaliculata. F is the sum of the daily food intake per snail within 15 days. W t is the terminal weight per snail. WGR, FR, FCR and SGR represent weight growth rate, feeding rate, food conversion ratio and specific growth rate, respectively. Different lowercase letters in the same column indicate significant differences between/among treatment groups of P. canaliculata of the same type at the 0.05 level.

Discussion
This study found that N. tabacum has great molluscicidal activity against P. canaliculata, and the survival rate of P. canaliculata decreased with increasing concentration of tobacco extract. The results confirm previous findings that tobacco wastes can be used as potential molluscicides against aquatic snails, and the wastes were not confined to leaves but also included tobacco dust, cigarette processing wastes and smoked filtered cigarette butts [20][21][22][23] . Whichever tobacco wastes are used, nicotine is the primary toxicant for controlling snails. A previous study used tobacco waste with 1.9 ppm nicotine at a rate of 1562.5 kg/ha to control P. canaliculata 11 . The nicotine content of the tobacco leaves used in this study was 1.1%, which is 5000 times higher than that in the cited study, and nearly 100% of the snails died in 2 g/L tobacco aqueous extract within 4 days. If the water in paddy field keeps at 3 cm deep and 2 g/L tobacco extract is equivalent to 600 kg/ha. Therefore, the amount of tobacco waste used and related labour costs will be greatly reduced. In fact, this study showed that tobacco aqueous extract effectively controlled P. canaliculata at 0.5 g/L and superiorly at 1 g/L. If ethanol is used instead of water for extracting tobacco, this will lead to a better inhibitory effect and lower usage 24 . Previous research showed that all apple snails (P. maculata) died within 48 h under 0.5 g/L ethanol extract of tobacco leaf, and the LC 50 was 0.38 g/L 23 . However, aqueous extracts have the advantages of low monetary cost and technological simplicity, and the tobacco waste can be returned directly to the field. According to our investigation, the lethal effect on P. canaliculata under tobacco extract stress was likely inversely linked to individual size because a higher proportion of adult snails died than juveniles (small and medium-sized), moreover, the LC 50 of adult snails was lower than that of the juveniles and smaller LC 50 values represented greater molluscicidal activity, which is an unusual observation in P. canaliculata, since larger individuals (< 40 mm in shell height) always showed higher survivability either in botanical extracts or chemical molluscicides 3,25,26 . Such a response was consistent with that in nitrogen-fertilized fields 27 . The reason for our discovery might be related to oxygen deficiency; the tobacco organic matter decomposed by microorganisms consumed dissolved oxygen and likely formed an anaerobic environment 28 . In addition, with the increasing concentration of tobacco extract, an oil slick formed that aggravated oxygen consumption. Combined with the fact that oxygen consumption shows a positive relationship with snail size 29 , this may have caused the snails to build up an oxygen debt that worsened with water fouling and poisoning caused by the tobacco extract 30 . Thus, younger snails may be more resistant to tobacco extract, and numerous studies claim that nicotine, as the principal tobacco alkaloid, could be rapidly absorbed by the respiratory system, resulting in cell proliferation and inhibition of apoptosis or even death 31 . However, the lethal effect of oxygen deficiency on P. canaliculata in this study still deserves further consideration since a larger space was provided for larger snails. Another possible explanation is that some adult snails might be reaching an advanced age, leading to decreased enzymatic activity and decreased chemical metabolic function 25 . Adults are a high priority in the prevention and control of P. canaliculata considering their high food intake and reproductive capacity 17 , and our discovery may be useful for the efficient control of P. canaliculata.
In addition to individual size, the sex ratio is another key factor in determining the extent of damage that P. canaliculata causes to rice and other aquatic crops 32 . Our study found that females exhibited greater viability than males when exposed to tobacco extract, and may have benefited from their own greater antioxidant capacity. Carotenoids are rich in the albumen gland of female P. canaliculata and have strong antioxidant activity 33 , which may reduce the damage caused by tobacco extract to a greater extent than in males. Previous studies have shown that females are more adaptable to cold, heat, drought and fasting than males, and females also grow faster and live longer than males 18,32 . These sex differences indicate that female P. canaliculata have higher stress resistance than males. However, from the perspective of survival time, mated females may suffer similar noxious effects of oxygen deficiency as males 30 . In addition, female P. canaliculata tend to preserve high energy reserves to achieve higher reproductive success, which is conducive to minimizing the impact of adversity. Males are Table 2. Effect of tobacco extract on the antioxidant enzyme and AChE activities of P. canaliculata. Different lowercase letters in the same column indicate significant differences between treatment groups of P. canaliculata of the same type at the 0.05 level. www.nature.com/scientificreports/ more susceptible to lethal or sublethal factors under adverse circumstances based on lower energy reserves and higher investment in mate-searching activity 34 .
In addition to the survival of P. canaliculata, the behaviour of the snails was significantly affected by tobacco extract. The snails nearly abandoned the chance to feed or mate in 0.5 g/L tobacco extract, and most stayed motionless or with a closed operculum before death; in response, the food intake dropped to below 3.5% of that in the control group. With a reduction in tobacco extract pressure, some snails exhibited feeding and crawling behaviours in 0.2 g/L tobacco extract, but reproduction was still greatly limited such that mating behaviour was observed only on the first day and no spawning occurred during the experimental period. Such a behavioural response was thought to be a strategy for attempting to survive and prosper until favourable conditions reappear 35 , overall, the survival, feeding and reproduction were severely limited under tobacco extract and the extent of inhibition is related to tobacco extract concentration, which indicates that tobacco extract has a marked control effect on the population development of P. canaliculata. The snails became inactive under tobacco extract stress likely because they were experiencing double harm from nicotine poisoning and oxygen deficiency. A previous study found that in another freshwater snail, Melanoides tuberculatus (Muller 1774), movement gradually decreased with increasing concentrations of tobacco waste, which was probably due to irritation from increased nicotine content 21 . In addition, oxygen restriction negatively affected activity in P. canaliculata, and active behaviours could further deplete oxygen 30 .
To survive in water environments containing tobacco waste, aquatic animals consume more energy to prevent physiological damage under inadequate feeding conditions, leading to a reduction in the energy available for growth 36,37 . In this study, the food intake and FR of different-sized P. canaliculata dropped to varying degrees under tobacco extract stress, which is possibly a key factor limiting growth performance; in fact, the growth of juvenile P. canaliculata was also restricted. Combined with the behaviours displayed by the snails in the tobacco extracts, we speculate that the snails needed energy intake to cope with nicotine poisoning and oxygen deficiency but did not receive sufficient energy to replenish this loss, which may have contributed to their low growth and eventual death 38 . The FCR is inversely proportional to body size in P. canaliculata 18 , but the FCR of juvenile snails under tobacco extract stress was below 90% of that in the control group with a smaller size. This phenomenon embodies a trade-off between physiological damage and reduced growth performance. Remarkably, the growth of adult P. canaliculata was unaffected by the tobacco extract, driven mainly by an increase in FCR. The potential causes likely included the females maintaining a high survival rate in 0.2 g/L tobacco extract, the females being mildly influenced by low concentrations of tobacco extract based on their strong stress resistance, and even a phenomenon of compensatory growth via increased FCR under tobacco stimulation. Previous research found that P. canaliculata snails exhibited compensatory growth by enhancing FR and FCR to protect them from intermittent drought stress 39 . In addition, females are often heavier than males under the same shell height condition 40 , which was reflected in our study. Mass mortality in males under tobacco stress indirectly led to an increase in the average weight of surviving individuals, which increased the feeding and growth data values in a somewhat disguised way.
The hepatopancreas is the key organ of bioaccumulation and detoxification of toxic substances in P. canaliculata 41,42 . Various antioxidant enzyme and AChE activity responses in the hepatopancreas have been observed under tobacco extract. Nicotine promotes reactive oxygen species (ROS) generation in a dose-dependent manner 43 , and SOD and CAT are dedicated to removing damaging ROS and are considered the first line of defence against lipid peroxidation and oxidative stress 44,45 . We found that SOD activity was increased by tobacco extract in medium and adult male snails, while a marked increase in CAT activity only occurred in adult males. GSH plays a role as a ROS scavenger and substrate in the antioxidant enzyme system 44 , and the elevation of GSH content in medium and adult male snails was found, which might help them reduce oxidative damage caused by tobacco extract. MDA is the metabolic product of lipid peroxidation and is regarded as a diagnostic indicator of oxidative tissue damage 5 . An increased MDA content was found in adult male P. canaliculata after exposure to tobacco extract, which means that the hepatopancreas tissue suffered oxidative damage or physiological injury and may further contribute to snail death. AChE is the primary neurotransmitter in the nervous system of animals and functions by hydrolyzing Ach 5 . In this study, AChE activity was significantly increased by tobacco extract in adult male P. canaliculata, and a similar phenomenon has been observed in rats, which may be related to the increase in ACh receptor ligands under nicotine stimulation 43,46 . ACh may be hydrolysed faster by excessive AChE activity 5 , and ACh receptor occupancy by nicotine results in improper ACh conduction 9 , leading to nervous dysfunction and blocked muscle movement, which might contribute to snail death 5 . Synthesizing the above results, it appeared that the responses of these typical antioxidases are size-and sex-dependent, and the antioxidant system of small and adult female P. canaliculata might not be activated under low concentrations (0.2 g/L) of tobacco extract, which is consistent with the survival of the snails and the survival advantages of these snails in response to tobacco exposure as described above.
This study comprehensively analysed the molluscicidal activities of tobacco extracts on P. canaliculata by measuring the survival, behaviour, feeding, growth, antioxidant enzyme and AChE activities of the snails, which indicated that discarded tobacco leaf has a strong molluscicidal activity and may have potential for use in the population management of P. canaliculata. Nevertheless, further field work is warranted to confirm the molluscicidal activity of tobacco extract on P. canaliculata in the context of complex ecosystems and to determine whether the lethal and sublethal effects of tobacco extract will be weakened by soil or other components in the field. The molluscicidal activities of extracts from different parts of discarded tobacco on P. canaliculata and the effects on rice or other crops also need to be further evaluated. It is remarkable that 1 g/L powdered tobacco leaves caused high mortality (> 80%) to adult P. canaliculata after introduced to simulated paddy field within 96 h, and promoted the growth of rice seedlings. But some snails could nibble on rice seedlings under 0.5 g/L powdered tobacco leaves. It's probably better approach that around 1 g/L powdered tobacco leaves is introduced to rice field 2-4 days before transplanting based on our experience, but needs further research and summarization. www.nature.com/scientificreports/

Conclusions
Our results showed that tobacco extract induced acute toxicity to P. canaliculata snails over 1 g/L, and molluscicidal activity progressively increased with the extract concentration and exposure time. Regardless of sex, the survival rate of P. canaliculata under tobacco extract stress was inversely proportional to its size, and adult males were more susceptible to the tobacco extract than females. The snails nearly abandoned the chance to feed or mate in 0.5 g/L tobacco extract, and reproduction was greatly limited at low concentrations (0.2 g/L). Longterm exposure to 0.2 g/L tobacco extract inhibited food intake in larger snails (> 15 mm shell height) and limited growth in juveniles (< 25 mm shell height). The antioxidant enzyme and AChE activities in the hepatopancreas of P. canaliculata in response to tobacco extract were size-and sex-dependent. The activities of SOD, CAT, AChE and the contents of GSH and MDA were increased in adult males after exposure to tobacco extract for 15 days, and increased SOD activity and GSH content were found in medium-sized snails (15-25 mm shell height). These results suggest that discarded tobacco leaves can be useful as a molluscicide for controlling the invasive snail P. canaliculata based on its effects on survival, behaviour, food intake, growth performance and antioxidant capacity.

Data availability
The datasets used and/or analyzed during the current study are included in this published article [and its supplementary information files]. www.nature.com/scientificreports/